Phenotypic and genotypic characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis clinical isolates in Hangzhou,China

ObjectivesPyrazinamide (PZA) is a crucial first-line tuberculosis (TB) drug recommended for both drug-susceptible and multidrug-resistant Mycobacterium tuberculosis. This study aimed to evaluate the performance of the sequencing method of pncA, rpsA and panD mutations in detecting PZA resistance in multidrug-resistant (MDR) TB isolates.MethodsWe sequenced the pncA, rpsA and panD genes and performed PZA susceptibility tests across 291 MDR-TB isolates to evaluate the performance of the sequencing method of these genes in detecting PZA resistance.ResultsResults showed that 145 (90.0%) of 161 PZA phenotypic resistant isolates had mutations in pncA. Among the 16 isolates (10.0%) which did not have mutations in pncA, ten and three isolates had mutations in rpsA and panD, respectively. The sequencing method for detecting mutations in pncA alone had 90.1% (95% confidence interval (CI), 84.4–94.2) sensitivity and 92.3% (95% CI, 86.3–96.3) specificity. The combination of all three genes increased the sensitivity from 90.1% (95% CI, 84.4–94.2) to 98.1% (95% CI, 94.7–99.6) (p < 0.001) while the specificity remained unchanged. In 120 PZA-susceptible and 16 PZA-resistant isolates without pncA mutations, rpsA/panD mutations were correlated with PZA resistance.ConclusionsPZA resistance was largely associated with mutations in pncA. Mutations in rpsA and panD were also associated with PZA resistance in MDR isolates expressing wild-type pncA. The detection of mutations in pncA, rpsA and panD can be useful for the determination of PZA resistance.     

Prevalence,associated factors,outcomes and transmission of extensively drug-resistant tuberculosis among multidrug-resistant tuberculosis patients in São Paulo,Brazil: a cross-sectional study

ObjectivesTo describe the prevalence, associated factors, treatment outcomes and transmission of extensively drug-resistant (XDR) tuberculosis (TB) in the state of São Paulo, Brazil, for 2011 to 2013.MethodsDrug susceptibility testing to first- and second-line drugs was performed by BACTEC MGIT 960 and molecular typing, by IS6110 restriction fragment length polymorphism. Clinical, epidemiologic and demographic data were obtained from surveillance information systems for TB. Patients were divided into three groups: multidrug resistant (MDR) TB (resistance to at least isoniazid and rifampicin), pre–XDR-TB (MDR-TB resistant to a fluoroquinolone or to at least one of the second-line injectable drugs) and XDR-TB (MDR-TB resistant to a fluoroquinolone and to at least one of the second-line injectables).ResultsAmong the 313 MDR-TB patients identified, the prevalence of XDR-TB and pre–XDR-TB was 10.2% (n = 32) and 19.2% (n = 60), respectively. Compared to MDR-TB patients, XDR-TB patients were more likely to be female (odds ratio (OR) = 2.74, 95% confidence interval (CI), 1.29–5.83), have a history of TB (OR = 5.16; 95% CI, 1.52–17.51) and present higher death rates (OR= 3.74; 95% CI 1.70–8.25). XDR-TB transmission was observed in households, between neighbours and between a patient and a healthcare worker in a hospital.ConclusionsThe prevalence of XDR-TB in the state of São Paulo is close to that estimated globally. Most of the XDR-TB patients were treated previously for TB and presented the lowest successful outcome rates. Because transmission of XDR-TB occurred, it is important that timely diagnosis of drug resistance is performed.     

Emergence of additional drug resistance during treatment of multidrug-resistant tuberculosis in China: a prospective cohort study

ObjectivesLittle is known about how additional second-line drug resistance emerges during multidrug-resistant tuberculosis (MDR-TB) treatment. The present study aimed to investigate the influence of microevolution, exogenous reinfection and mixed infection on second-line drug resistance during the recommended 2-year MDR-TB treatment.MethodsIndividuals with MDR-TB were enrolled between 2013 and 2016 in a multicentre prospective observational cohort study and were followed up for 2 years until treatment completion. Whole-genome sequencing (WGS) was applied for serial Mycobacterium tuberculosis isolates from study participants throughout the treatment, to study the role of microevolution, exogenous reinfection and mixed infection in the development of second-line drug resistance.ResultsOf the 286 enrolled patients with MDR-TB, 63 (22.0%) M. tuberculosis isolates developed additional drug resistance during the MDR-TB treatment, including 5 that fulfilled the criteria of extensively drug-resistant TB. By comparing WGS data of serial isolates retrieved from the patients throughout treatment, 41 (65.1%) of the cases of additional second-line drug resistance were the result of exogenous reinfection, 18 (28.6%) were caused by acquired drug resistance, i.e. microevolution, while the remaining 4 (6.3%) were caused by mixed infections with drug-resistant and drug-susceptible strains. In multivariate analysis, previous TB treatment (adjusted hazard ratio (aHR) 2.51, 95% CI 1.51–4.18), extensive disease on chest X-ray (aHR 3.39, 95% CI 2.03–5.66) and type 2 diabetes mellitus (aHR 4.00, 95% CI 2.22–7.21) were independent risk factors associated with the development of additional second-line drug resistance.ConclusionsA large proportion of additional second-line drug resistance emerging during MDR-TB treatment was attributed to exogenous reinfection, indicating the urgency of infection control in health facilities as well as the need for repeated drug susceptibility testing throughout MDR-TB treatment.     

Antimicrobial resistance of Helicobacter pylori in Germany, 2015 to 2018

ObjectivesNational and international guidelines recommend empiric first-line treatments of individuals infected with Helicobacter pylori without prior antimicrobial susceptibility testing. For this reason, knowledge of primary resistance to first-line antibiotics such as clarithromycin is essential. We assessed the primary resistance of H. pylori in Germany to key antibiotics by molecular genetic methods and evaluated risk factors for the development of resistance.MethodsGastric tissue samples of 1851 yet treatment-naïve H. pylori-positive patients were examined with real-time PCR or PCR and Sanger sequencing for mutations conferring resistance to clarithromycin, levofloxacin and tetracycline. Clinical and epidemiological data were documented and univariable and multivariable logistic regression analyses were conducted.ResultsOverall primary resistances were 11.3% (210/1851) to clarithromycin, and 13.4% (201/1497) to levofloxacin; resistance to tetracycline (2.5%, 38/1497) was as low as combined resistance to clarithromycin/levofloxacin (2.6%, 39/1497). Female sex and prior antimicrobial therapies owing to unrelated bacterial infections were risk factors for clarithromycin resistance (adjusted OR (aOR) 2.3, 95% CI 1.6–3.4; and 2.6, 95% CI 1.5–4.5, respectively); older age was associated with levofloxacin resistance (aOR for those ≥65 years compared with those 18–35 years: 6.6, 95% CI 3.1–14.2).ConclusionsClarithromycin might still be recommended in first-line eradication therapies in yet untreated patients, but as nearly every tenth patient may carry clarithromycin-resistant H. pylori it may be advisable to rule out resistance ahead of treatment by carrying out susceptibility testing or prescribing an alternative therapy.     

Pyrazinamide resistance among South African multidrug-resistant Mycobacterium tuberculosis isolates

Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.     

Bacillus Calmette–Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control

ObjectiveLaboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette–Guérin (BCG)).MethodsSeven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC₉₀. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently.ResultsOur panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance.ConclusionBCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.     

Current in-hospital management for patients with tuberculosis in a high-income country: a retrospective cohort study

ObjectivesIn Japan, most cases of tuberculosis (TB) occur among individuals aged 65 years or older. However, data on in-hospital adverse events (AEs) associated with TB management, especially in high-income nations with an ageing population, are scarce. The present study aimed to scrutinize the current TB unit practices, incidence of in-hospital AEs and predictors of in-hospital mortality.MethodsThis retrospective cohort study was conducted at a tertiary care centre in Tokyo, Japan from 2012 to 2017. Inpatients with the diagnosis of TB and aged >18 years were included. Quality of in-hospital care and factors associated with in-hospital mortality were investigated using multivariate logistic regression analysis.ResultsIn total, 448 patients were enrolled. The in-hospital mortality rate was 16.7% (75/448). Miliary/disseminated TB was common (59/448, 13.2%), especially in those who died (17/75, 22.7%). Factors independently associated with in-hospital mortality were a low Karnofsky performance status score on admission (score: 40-10, adjusted odds ratio (aOR) 25.65, 95% CI 5.63–116.92 and score: 70-50, aOR 9.47, 95% CI 2.07–43.3), age over 89 years (aOR 3.68, 95% CI 1.08–12.46), Charlson Co-morbidity Index >5 (aOR 3.56, 95% CI 1.37–9.21), development of any health-care-associated infection (aOR 2.95, 95% CI 1.35–6.41), and development of any drug-related AE leading to discontinuation of anti-TB agents (seven patients were unable to resume treatment with anti-TB agents before death) (aOR 2.29, 95% CI 1.02–5.11).ConclusionsIn-hospital AEs (i.e. health-care-associated infection and drug-related AEs), as well as patient-related variables, were associated with in-hospital mortality among TB patients.     

The time-varying association between previous antibiotic use and antibiotic resistance

ObjectivesThe objective of the study was to estimate how the time elapsed from previous antibiotic use is associated with antibiotic resistance.MethodsData comprised electronic medical records of all patients in an Israeli hospital who had a positive bacterial culture from 2016 to 2019. These included susceptibility testing results and clinical and demographic data. Mixed-effects time-varying logistic models were fitted to estimate the association between the time elapsed since the last use of aminoglycosides and gentamicin resistance (n = 13 095), cephalosporins and ceftazidime resistance (n = 13 051), and fluoroquinolones and ciprofloxacin resistance (n = 15 364) while adjusting for multiple covariates.ResultsFor all examined antibiotics, previous antibiotic use had a statistically significant association with resistance (p < 0.001). These associations exhibited a clear decreasing pattern over time, which we present as a flexible function of time. Nonetheless, previous antibiotic use remained a significant risk factor for resistance for at least 180 days when the adjusted ORs were 1.94 (95% CI, 1.40–2.69), 1.33 (95% CI, 1.10–1.61), and 2.25 (95% CI, 1.49–3.41) for gentamicin, ceftazidime, and ciprofloxacin, respectively.DiscussionThe association between prior antibiotic use and resistance decreases over time. Commonly used cut-offs for prior antibiotic use can either misclassify patients still at higher risk when too recent or provide a diluted estimate of the effects of antibiotic use on future resistance when too distant. Hence, prior antibiotic use should be considered a time-dependent risk factor for resistance in both epidemiological research and clinical practice.     

A systematic review of Mycobacterium leprae DNA gyrase mutations and their impact on fluoroquinolone resistance

BackgroundThe fact that Mycobacterium leprae does not grow in vitro remains a challenge in the survey of its antimicrobial resistance (AMR). Mainly molecular methods are used to diagnose AMR in M. leprae to provide reliable data concerning mutations and their impact. Fluoroquinolones (FQs) are efficient for the treatment of leprosy and the main second-line drugs in case of multidrug resistance.ObjectivesThis study aimed at performing a systematic review (a) to characterize all DNA gyrase gene mutations described in clinical isolates of M. leprae, (b) to distinguish between those associated with FQ resistance or susceptibility and (c) to delineate a consensus numbering system for M. leprae GyrA and GyrB.Data sourcesData source was PubMed.Study eligibility criteriaPublications reporting genotypic susceptibility-testing methods and gyrase gene mutations in M. leprae clinical strains.ResultsIn 25 studies meeting our inclusion criteria, 2884 M. leprae isolates were analysed (2236 for gyrA only (77%) and 755 for both gyrA and gyrB (26%)): 3.8% of isolates had gyrA mutations (n = 110), mostly at position 91 (n = 75, 68%) and 0.8% gyrB mutations (n = 6). Since we found discrepancies regarding the location of substitutions associated with FQ resistance, we established a consensus numbering system to properly number the mutations. We also designed a 3D model of the M. leprae DNA gyrase to predict the impact of mutations whose role in FQ-susceptibility has not been demonstrated previously.ConclusionsMutations in DNA gyrase are observed in 4% of the M. leprae clinical isolates. To solve discrepancies among publications and to distinguish between mutations associated with FQ resistance or susceptibility, the consensus numbering system we proposed as well as the 3D model of the M. leprae gyrase for the evaluation of the impact of unknown mutations in FQ resistance, will provide help for resistance surveillance.     

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.     

Association of MDR-TB isolates with clinical characteristics of patients from Northern region of India

Purpose: We sought to determine the characteristics and relative frequency of transmission of MDR-TB in North India and their association with the clinical and epidemiological characteristics of TB-patients. Materials and Methods: To achieve the objectives PCR-SSCP, MAS-PCR and direct DNA sequencing were used against 101 Mycobacterium tuberculosis isolates. Results: Multidrug-resistant-TB isolates were found to be significantly higher (P = 0.000) in previously treated patients in comparison to newly diagnosed patients. Further, significant differences (P = 0.003) were observed between different age groups (Mean ± SD, 28.6 ± 11.77) of the TB patients and multidrug resistance. Most frequent mutations were observed at codons 531 and 315 of rpoB and katG genes, respectively, in MDR-TB isolates. Conclusion: Routine surveillance of resistance to anti-TB drugs will improve timely recognition of MDR-TB cases and help prevent further transmission in Northern India.     

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis

Background: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.     

Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR

Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.     

Prospective study on antimicrobial resistance in leprosy cases diagnosed in France from 2001 to 2015

Antimicrobial resistance (AMR) in leprosy is mostly unknown because Mycobacterium leprae does not grow in vitro and bacteriologic investigations have been abandoned. However, molecular detection of resistance can be applied to multibacillary cases. Patients living in France mainland or in the French territories and diagnosed with leprosy from 2001 to 2015 were prospectively studied for AMR by detecting mutations in rpoB for rifampicin resistance, in folP1 for dapsone and in gyrA for ofloxacin. Single nucleotide polymorphism (SNP) genotypes 1–4 were determined for resistant strains. Of 334 skin biopsy samples received for suspicion of leprosy, 184 (55.1%) were positive for M. leprae (acid-fast bacilli and M. leprae–specific repetitive element PCR) corresponding to 160 multibacillary cases. AMR was detected in 18 cases (11.3%): 13 cases (8.1%) of dapsone resistance, three (1.9%) rifampicin and two (1.3%) ofloxacin. There were no strains with multidrug resistance. The mutations (numbering system of M. leprae TN strain genome) found were P55L (n = 7), T53I (n = 5), T53A (n = 1) in folP1; S456L (n = 2) and S456F (n = 1) in rpoB; and A91V (n = 2) in gyrA. Resistance proportion differ significantly between new and relapse cases (9/127, 7.0%, vs. 9/33, 25.7%, p 0.003). The frequency distribution of SNP1–4 types of resistant strains was two, one, 12 and three with five SNP3 cases from New Caledonia harbouring the same T53I FolP1 substitution. This is the first report of AMR surveillance for new and relapse cases of leprosy in Europe. Detection of resistance helped in individual treatment as well as in epidemiologic investigations.     

Tuberculosis incidence and mortality in people living with human immunodeficiency virus: a Danish nationwide cohort study

ObjectivesTo explore changes over time in the epidemiology of tuberculosis (TB) in Denmark in people living with human immunodeficiency virus (HIV) (PLWH).MethodsIn this nationwide, population-based cohort study we included all adult PLWH from the Danish HIV Cohort Study (1995–2017) without previous TB. We estimated TB incidence rate (IR), all-cause mortality rate (MR), associated risk and prognostic factors using Poisson regression.ResultsAmong 6982 PLWH (73 596 person-years (PY)), we observed 217 TB events (IR 2.9/1000 PY, 95% CI 2.6–3.4: IR 6.7, 95% CI 5.7–7.9 among migrants and IR 1.4, 95% CI 1.1–1.7 among Danish-born individuals; p < 0.001). The IR of concomitant HIV/TB remained high and unchanged over time. The IR of TB diagnosed >3 months after HIV diagnosis declined with calendar time, longer time from HIV diagnosis, and CD4 cell recovery. Independent TB risk factors were African/Asian/Greenland origin (adjusted incidence rate ratio (aIRR) 5.2, 95% CI 3.5–7.6, aIRR 6.5, 95% CI 4.2–10.0, aIRR 7.0, 95% CI 3.4–14.6, respectively), illicit drug use (aIRR 6.9, 95% CI 4.2–11.2), CD4 <200 cells/μL (aIRR 2.7, 95% CI 2.0–3.6) and not receiving antiretroviral therapy (aIRR 3.7, 95% CI 2.5–5.3). Fifty-five patients died (MR 27.9/1000 PY, 95% CI 21.4–36.3), with no improvement in mortality over time. Mortality prognostic factors were Danish-origin (adjusted mortality rate ratio (aMRR) 2.3, 95% CI 1.3–4.3), social burden (aMRR 3.9, 95% CI 2.2–7.0), CD4 <100 cells/μL at TB diagnosis (aMRR 2.6, 95% CI 1.3–4.9), TB diagnosed >3 months after HIV versus concomitant diagnosis (aMRR 4.3, 95% CI 2.2–8.7) and disseminated TB (aMRR 3.3, 95% CI 1.1–9.9).ConclusionLate HIV presentation with concomitant TB remains a challenge. Declining TB rates in PLWH were observed over time and with CD4 recovery, highlighting the importance of early and successful antiretroviral therapy. However, MR remained high. Our findings highlight the importance of HIV and TB screening strategies and treatment of latent TB in high-risk groups.     

Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009–15

ObjectivesAntimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network.MethodsFrom 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA.ResultsAmong 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases.ConclusionsThis is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.     

Antibiotic resistance associated with the COVID-19 pandemic: a systematic review and meta-analysis

BackgroundCOVID-19 and antimicrobial resistance (AMR) are two intersecting global public health crises.ObjectiveWe aimed to describe the impact of the COVID-19 pandemic on AMR across health care settings.Data sourceA search was conducted in December 2021 in WHO COVID-19 Research Database with forward citation searching up to June 2022.Study eligibilityStudies evaluating the impact of COVID-19 on AMR in any population were included and influencing factors were extracted. Reporting of enhanced infection prevention and control and/or antimicrobial stewardship programs was noted.MethodsPooling was done separately for Gram-negative and Gram-positive organisms. Random-effects meta-analysis was performed.ResultsOf 6036 studies screened, 28 were included and 23 provided sufficient data for meta-analysis. The majority of studies focused on hospital settings (n = 25, 89%). The COVID-19 pandemic was not associated with a change in the incidence density (incidence rate ratio 0.99, 95% CI: 0.67–1.47) or proportion (risk ratio 0.91, 95% CI: 0.55–1.49) of methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci cases. A non-statistically significant increase was noted for resistant Gram-negative organisms (i.e. extended-spectrum beta-lactamase, carbapenem-resistant Enterobacterales, carbapenem or multi-drug resistant or carbapenem-resistant Pseudomonas aeruginosa or Acinetobacter baumannii, incidence rate ratio 1.64, 95% CI: 0.92–2.92; risk ratio 1.08, 95% CI: 0.91–1.29). The absence of reported enhanced infection prevention and control and/or antimicrobial stewardship programs initiatives was associated with an increase in gram-negative AMR (risk ratio 1.11, 95% CI: 1.03–1.20). However, a test for subgroup differences showed no statistically significant difference between the presence and absence of these initiatives (p 0.40).ConclusionThe COVID-19 pandemic may have hastened the emergence and transmission of AMR, particularly for Gram-negative organisms in hospital settings. But there is considerable heterogeneity in both the AMR metrics used and the rate of resistance reported across studies. These findings reinforce the need for strengthened infection prevention, antimicrobial stewardship, and AMR surveillance in the context of the COVID-19 pandemic.     

Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm

There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with pyrazinamide-resistant MDR-TB. We identified from the Dutch national database (2007-11) patients with a positive Mycobacterium tuberculosis culture containing a mutation in the rpoB gene. In these cases, we prospectively sequenced the pncA gene. Results from the rpoB and pncA mutation analysis (pncA added to rpoB) were compared with phenotypic susceptibility testing results to rifampicin, isoniazid and pyrazinamide (reference standard) using the Mycobacterial Growth Indicator Tube 960 system. We included 83 clinical M. tuberculosis isolates containing rpoB mutations in the primary analysis. Rifampicin resistance was seen in 72 isolates (87%), isoniazid resistance in 73 isolates (88%) and MDR-TB in 65 isolates (78%). Phenotypic reference testing identified pyrazinamide-resistant MDR-TB in 31 isolates (48%). Sensitivity of pncA sequencing added to rpoB mutation analysis for detecting pyrazinamide-resistant MDR-TB was 96.8%, the specificity was 94.2%, the positive predictive value was 90.9%, the negative predictive value was 98.0%, the positive likelihood was 16.8 and the negative likelihood was 0.03. In conclusion, pyrazinamide-resistant MDR-TB can be accurately detected using pncA sequencing added to rpoB mutation analysis. We propose to include pncA sequencing in every isolate with an rpoB mutation, allowing for stratification of MDR-TB treatment according to pyrazinamide susceptibility.     

Predictors for treatment outcomes among patients with drug-susceptible tuberculosis in the Netherlands: a retrospective cohort study

ObjectivesWe evaluated treatment outcomes and predictors for poor treatment outcomes for tuberculosis (TB) among native- and foreign-born patients with drug-susceptible TB (DSTB) in the Netherlands.MethodsThis retrospective cohort study included adult patients with DSTB treated from 2005 to 2015 from a nationwide exhaustive registry. Predictors for unsuccessful treatment outcomes (default and failure) and TB-associated mortality were analysed using multivariate logistic regression.ResultsAmong 5674 identified cases, the cumulative incidence of unsuccessful treatment and mortality were 2.6% (n/N = 146/5674) and 2.0% (112/5674), respectively. Although most patients were foreign-born (71%; 4042/5674), no significant differences in these outcomes were observed between native- and foreign-born patients (p > 0.05). Significant predictors for unsuccessful treatment were aged 18–24 years (odds ratio (OR), 2.04; 95% CI 1.34–3.10), homelessness (OR, 2.56; 95% CI 1.16–5.63), prisoner status (OR, 5.39; 95% CI 2.90–10.05) and diabetes (OR, 2.02; 95% CI 1.03–3.97). Furthermore, predictors for mortality were aged 74–84 years (OR, 5.58; 95% CI 3.10–10.03) or ≥85 years (OR, 9.35, 95% CI 4.31–20.30), combined pulmonary and extra-pulmonary TB (OR, 4.97; 95% CI 1.42–17.41), central nervous system (OR, 120, 95% CI 34.43–418.54) or miliary TB (OR, 10.73, 95% CI 2.50–46.02), drug addiction (OR, 3.56; 95% CI 1.34–9.47) and renal insufficiency/dialysis (OR, 3.23; 95% CI 1.17–8.96).ConclusionsNative- and foreign-born patients exhibited similar TB treatment outcomes. To further reduce disease transmission and inhibit drug resistance, special attention should be given to high-risk patients.     

Resistance Sniffer: An online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data

The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up.ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.